The present invention relates to the field of genetic engineering. More particularly, the invention relates to a novel method for assembling two or more DNA fragments with high efficiency and yield.
Building DNA constructs is the basis of genetic engineering. Manipulating recombinant DNA and its incorporation into vectors is common knowledge in the art. For example, fragments obtained by PCR techniques, may be incorporated into plasmids for cloning purposes. Generating specific DNA constructs by joining together different DNA segments, each encoding specific properties and/or possessing specific functions, is also known in the art. The state of the art has been published in a very large number of books, articles, patent applications, patents and the like, and is usually readily available and known to all of skill in the art. For example, a comprehensive account of DNA cloning procedures is provided in the three volume text by Sambrook et al. (1989) entitled xe2x80x9cMolecular Cloning-a Laboratory Manualxe2x80x9d 2nd edition, Cold Spring Harbor Laboratory Press. This extensive account of the prior art techniques for the combination of DNA fragments, cloning and expression thereof, is included herein by reference, in its entirety.
According to the present state of the art, the DNA fragments comprising the plasmid to be constructed are cleaved by restriction enzymes from larger entities. Next, they are connected utilizing the enzyme Ligase, which forms phosphodiester bonds between DNA fragments. Restriction enzymes cleave DNA leaving blunt or staggered ends (protruding short single stranded termini) also called overhangs. The efficiency of the ligation of blunt ended fragments is very low. Using staggered ended fragments elevates the efficiency of the ligation process, because the two fragments are being held together by the hydrogen-bonds between the complementary overhangs. Because the overhangs produced by most of the restriction enzymes are very short (2-4 bases), the connections between the complementary overhangs are weak and not very specific. Thus, when constructing a plasmid from restriction enzyme produced fragments, the yield is very low and there are many illegitimate ligations, leading to undesired products. It is therefore necessary to amplify the product usually by transfecting cells of choice such as bacterial cells, and then to identify and isolate colonies containing the DNA of choice from the ones containing undesired products. Only then can another DNA fragment be added by repetition of the whole procedure. As a result of this inefficient processes the building of complex DNA constructs is a laborious, time consuming and expensive process, in which each step necessitates the successful completion of the former. The construction of complex molecules may take several weeks to several months. Sometimes the completion of such constructs is not achieved at all. Another major drawback of the above mentioned method is that the availability of restriction site does not always coincide the construction demands. One way to overcome the above mentioned problems is to use short artificial DNA molecules called xe2x80x9clinkersxe2x80x9d. This however further complicates the construction process, reduces the yield, increases the percentage of wrong constructs and sometimes add undesired foreign sequences.
Several methods suggest the possibility of connecting DNA fragments by producing longer complementary overhangs (8-14 nucleotides). These connections are more stable than the ones produced by restriction enzymes. In fact, they are stable enough to render the ligation step unnecessary and the bacterial cells may be transformed right after the fragments are connected. The phosphodiester bonds are generated later, by the endogenous bacterial ligation machinery.
One prior method uses single strand extensions that are created by adding nucleotides at the 3xe2x80x2 end of a DNA strand in a template-independent fashion (Roychoudhury, R. Gene Amplif. Anal. 2:41-83, 1981). The enzyme used in this method, Terminal Transferase, incorporates nucleotides at the end of a double-stranded DNA fragment, thus creating a single-stranded tail. Since the enzyme uses the nucleotides randomly, the only way to ensure that the single-stranded tail will be complementary to a corresponding overhang created on a second DNA molecule, is to provide for each extension only one of the four nucleotides. The overhangs created with this method must therefore be homopolymeric, so that only four types of overhangs can be used, corresponding to the residues dA, dC, dG or dT. Since the overhangs created on both termini of a DNA fragment must be identical, cloning with this method is directionless and can only involve two fragments that are connected to each other at both ends, forming a circular molecule. Furthermore, the length of the overhangs cannot be specifically controlled. Finally, the method necessarily introduces an unwanted stretch of nucleotides into the final construct, the length of which cannot be determined exactly, making the method unsuitable for the purpose of cloning into expression vectors where the reading frame must be preserved.
According to another method (the commercial product xe2x80x9cPCR-Direct(trademark)xe2x80x9d, manufactured by CLONTECH, Inc., USA) the overhangs are generated utilizing the exonuclease activity of a DNA polymerase.
U.S. Pat. No. 5,137,814 describes another method in which the overhang is generated by providing at least one dU residue instead of dT, close to the terminus of the fragment. The position of the dU residues determines the length of the overhang. This method involves an a-purination of the Uracil bases. The a-purinated residues no longer have hydrogen-bond connections with their complementary bases on the opposite strand. Moreover, they destabilize the hydrogen bonds of their neighboring bases as well. The resulting 3xe2x80x2 protruding termini may connect to a complementary single strand sequence. A commercial product using this method is the CloneAmp(copyright) pAMP1 System (manufactured by Life Technologies Inc., USA). In the aforesaid method a 12-base overhang is used.
The use of overhangs longer than 4 nucleotides for the purpose of fragment cloning is also described in several other publications. Rashtchian et al. (Anal. Biochem. 206, p. 91-97, 1992), describe 12 nucleotide overhangs generated by Uracil DNA Glycosylase (UDG) to achieve high-efficiency cloning of single inserts into a vector. Kuijper et al. (Gene 112, p. 147-155, 1992) and Aslanidis et al. (PCR Methods Appl. 4:172-177, 1994), describe a cloning method wherein T4 polymerase is used together with a predetermined dNTP to generate overhangs of a certain length in PCR products. This method requires a specific sequence to be present in the PCR primer. Hsiao et al. (Nucleic Acids Res. 21, p. 5528-5529, 1993) and Yang et al. (Nucleic Acids Res. 21, 1889-1893, 1993) disclose generation of overhangs by the exonucleolytic activity of Exonuclease III (Exo III) or of T4 polymerase. Overhangs of 12 (Aslanidis), 8 (Yang) and 10-14 (Hsiao) nucleotides are disclosed.
The probability of producing a joint molecule composed from three molecules is the probability of joining of the first two molecules, multiplied by the probability of joining of the second and third molecules. If the joining of any two molecule is a rare event, due to the relatively short overhang, the joining of three or more molecules becomes practically useless for cloning purposes, because of the resulting low efficiency
It is therefore clear that the present state of the art is not satisfactory and there is a need for an improved method by which several DNA fragments can be effectively joined together in a directional, predetermined way, in one single-step. Such a method will remove the limitations imposed on genetic engineering by the aforementioned methods, and will thus revolutionize the way in which genetic engineering is performed.
In one aspect the invention is directed to a method for assembling two or more DNA fragments with high efficiency, comprising:
a) providing, for each DNA fragment, at least one protruding terminus, or xe2x80x9coverhangxe2x80x9d, capable of hydrogen bonding to a complementary sequence on at least one strand of a second DNA fragment, said overhang having at least 15 bases; and
b) mixing two or more said DNA fragments under conditions suitable to promote joining thereof.
The method of the invention is based on the very surprising finding that increasing the number of bases in complementary overhang from 12 to at least 15, permits to reduce the ratio of the reagents from about 1:100-300 to about 1:1. At the same time, this permits to join multiple fragments (3 or more), because of the high efficiency of the process.
According to one embodiment of the invention three or more fragments are joined by the method of the invention, and the molar ratio of each DNA pairs is about 1:1 to 1:50.
The present invention provides a DNA fragment comprising an overhang of at least 15 nucleotides or an end portion suitable to be converted into such an overhang. The invention further provides said DNA fragment, for use in the above method of the invention.
According to another preferred embodiment of the invention, the number of bases in the overhang is between 20 and 30. While, as stated above, the improvement of the process efficacy is very dramatic when passing from 12 to 15 bases, there is still a further steep improvement in the efficacy, when increasing the number of bases to about 20. This increase reaches a plateau above 20 bases. Taking into account that, when joining a number of fragments, the overall efficiency of the process decreases, it is of course desirable to employ the lowest number of bases in the overhang which still provides for increased joining efficiency. As stated, in most cases this optimal length will be in the neighborhood of 20 bases.
As will be appreciated by the skilled person, this is most surprising because such a dramatic increase in binding ability of fragments (from almost no binding when overhangs are 12 bases long to almost 100% binding when the overhangs are 21 bases long) was unexpectable. Although some improvement could have been expected by increasing the length of the overhang, such a dramatic improvement (two orders of magnitude) was entirely surprising. The art, in fact, has continued to use stepwise cloning techniques, with all the inherent disadvantages, and has not attempted in practice to use overhangs longer than 12 bases.